US20230159632A1 - Anti-il-17a antibody and use thereof - Google Patents

Anti-il-17a antibody and use thereof Download PDF

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US20230159632A1
US20230159632A1 US17/413,152 US201917413152A US2023159632A1 US 20230159632 A1 US20230159632 A1 US 20230159632A1 US 201917413152 A US201917413152 A US 201917413152A US 2023159632 A1 US2023159632 A1 US 2023159632A1
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antibody
antigen
binding fragment
amino acid
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Jian Yao
Dan Meng
Hui Feng
Sheng Yao
Hai Wu
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to an antibody and an antigen-binding fragment thereof that specifically bind to IL-17A.
  • the present invention particularly relates to antibodies and antigen-binding fragments thereof that inhibit IL-17A-mediated bioactivity, and compositions containing the antibodies or antigen-binding fragments, and methods for the treatment of related diseases.
  • the present invention more particularly relates to use of anti-IL-17A antibodies and antigen-binding fragments thereof in the treatment of immune pathological diseases, including autoimmune and inflammatory diseases such as rheumatoid arthritis, psoriasis, ankylosing spondylitis, multiple sclerosis, systemic lupus erythematosus (SLE), lupus nephritis, or chronic obstructive pulmonary disease, asthma, infectious granuloma, cystic fibrosis, or cancer.
  • autoimmune and inflammatory diseases such as rheumatoid arthritis, psoriasis, ankylosing spondylitis, multiple sclerosis, systemic lupus erythematosus (SLE), lupus nephritis, or chronic obstructive pulmonary disease, asthma, infectious granuloma, cystic fibrosis, or cancer.
  • Interleukin 17 also known as CTLA-8 or IL-17A, plays a key role in the immune system.
  • the IL-17 family includes six members, i.e., IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, all of which contain 4 highly conserved cysteine residues that are crucial.
  • IL-17A and IL-17F have the most similar homology and biological functions, and are studied most deeply at present.
  • IL-17A expressed in vivo has an N-terminal signal peptide consisting of 23 amino acids, which is cleaved to generate mature IL-17A.
  • IL-17A is disulfide-linked, and is typically secreted and present as a homodimer. Sometimes, it also binds to IL-17F to form a heterodimer IL-17AF.
  • IL-17A or IL-17 refers to IL-17A homodimer protein which is produced predominantly by helper T-cells 17 (T helper 17 or Th17), and may also be synthesized and secreted by other immune cells such as ⁇ T cells, lymphoid tissue indicator (LTi) cells, innate lymphoid cells (ILCs) and natural killer T (NKT) cells (Cua D J, Tato C M., Nature reviews. 2010, 10:479-489).
  • T helper 17 or Th17 helper T helper 17
  • LTi lymphoid tissue indicator
  • ILCs innate lymphoid cells
  • NKT natural killer T
  • IL-17A when present, causes an inflammatory outbreak and tissue damage by promoting the stability of Th17 cells for their continuous secretion of IL-17A, up-regulating the expression of pro-inflammatory factors (IL-22, CSF-2 and IFN- ⁇ ) and down-regulating the expression of anti-inflammatory factors (IL-2, IL-27 and IL-12) and other ways (McGeachy M J, et al., Nature immunology. 2009, 10:314-324). Therefore, the IL-17A pathway plays a critical role in tissue damage when IL-23 is abnormally expressed in tissues.
  • IL-17 is generally secreted at a specific site and acts in local tissues by binding to the IL-17 receptor (IL-17R) on the surface of target cells.
  • the IL-17R family includes five members, that is IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE, which are widely expressed on various cell membranes (Iwakura Y, et al., Immunity. 2011; 34:149-162).
  • IL-17 mainly exerts effects by binding to IL-17RA/IL-17RC complex on the surface of cells of non-hematopoietic origin (such as epithelial cells and mesenchymal cells) (Ishigame H, et al., Immunity.
  • cytokines such as IL-6, G-CSF, GM-CSF, IL-10, TGF- ⁇ and TNF- ⁇
  • chemokines including IL-8, CXCL1 and MCP-1
  • prostaglandin e.g., PGE2
  • IL-17 Autoimmune diseases, such as psoriasis, rheumatoid arthritis, ankylosing spondylitis, Crohn's disease and multiple sclerosis, pose a serious threat to human health. It was found that the dyssecretosis of IL-17 is closely related to the occurrence and development of such diseases. Antibodies targeting IL-17 are effective in alleviating symptoms of autoimmune diseases by inhibiting the IL-17-IL-17R signaling pathway (Sarah L, et al., Nat Rev Immunol. 2014, 14(9): 585-600). Cosentyx (secukinumab), developed by Novartis, is the first IL-17 monoclonal antibody in the world.
  • the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human IL-17A, comprising at least one complementarity determining region (CDR) sequence selected from SEQ ID NOs: 1-24 and 60-65.
  • CDR complementarity determining region
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises at least one heavy chain CDR domain selected from SEQ ID NOs: 1-3, 4-6, 7-9, 10-12 and 60-62, and/or at least one light chain CDR domain selected from SEQ ID NOs: 13-15, 16-18, 19-21, 22-24 and 63-65.
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH), wherein the VH comprises an HCDR1 selected from SEQ ID NOs: 1, 4, 7, 10 and 60, an HCDR2 selected from SEQ ID NOs: 2, 5, 8, 11 and 61, and an HCDR3 selected from SEQ ID NOs: 3, 6, 9, 12 and 62.
  • VH heavy chain variable region
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH), wherein the VH comprises a set of HCDR1, HCDR2 and HCDR3 amino acid sequences selected from any of sets A to E:
  • VH heavy chain variable region
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a light chain variable region (VL), wherein the VL comprises an LCDR selected from SEQ ID NOs: 13, 16, 19, 22 and 63, an LCDR2 selected from SEQ ID NOs: 14, 17, 20, 23 and 64, and an LCDR3 selected from SEQ ID NOs: 15, 18, 21, 24 and 65.
  • VL light chain variable region
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a light chain variable region (VL), wherein the VL comprises a set of LCDR1, LCDR2 and LCDR3 amino acid sequences selected from any of sets F to J:
  • VL light chain variable region
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the variable regions comprise a set of 6 CDR amino acid sequences selected from any of sets I to VI:
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the VH has an amino acid selected from SEQ ID NOs: 25, 26, 27, 28, 33, 35, 38 and 40, and/or the VL has an amino acid sequence selected from SEQ ID NOs: 29, 30, 31, 32, 34, 36, 38, 39 and 41.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the variable regions have a set of amino acid sequences selected from any of sets 1 to 7:
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a light chain (LC) and/or a heavy chain (HC), wherein the HC has an amino acid sequence selected from SEQ ID NOs: 42, 44, 46 and 49, and/or the LC has an amino acid sequence selected from SEQ ID NOs: 43, 45, 47, 48 and 50.
  • LC light chain
  • HC heavy chain
  • the HC has an amino acid sequence selected from SEQ ID NOs: 42, 44, 46 and 49
  • the LC has an amino acid sequence selected from SEQ ID NOs: 43, 45, 47, 48 and 50.
  • the antibody or the antigen-binding fragment thereof disclosed herein comprises a light chain (LC) and a heavy chain (HC), wherein the LC has an amino acid sequence set forth in SEQ ID NO: 43, and the HC has an amino acid sequence set forth in SEQ ID NO: 42 or 44; the LC has an amino acid sequence set forth in SEQ ID NO: 45, and the HC has an amino acid sequence set forth in SEQ ID NO: 44; the LC has an amino acid sequence set forth in SEQ ID NO. 47 or 48, and the HC has an amino acid sequence set forth in SEQ ID NO: 46; or the LC has an amino acid set forth in SEQ ID NO: 50, and the HC has an amino acid sequence set forth in SEQ ID NO: 49.
  • the LC has an amino acid sequence set forth in SEQ ID NO: 43
  • the HC has an amino acid sequence set forth in SEQ ID NO: 42 or 44
  • the LC has an amino acid sequence set forth in SEQ ID NO: 45
  • the HC has an amino acid sequence set
  • the antibody disclosed herein is an intact antibody, preferably IgG, and more preferably IgG4.
  • the antibody disclosed herein is 1F8, 2B2, 2F5, ch1, ch2, ch16, hu31, hu43, hu44, hu59, hu60 or hu250.
  • the antibody or the antigen-binding fragment thereof disclosed herein is characterized by: a) specifically binding to IL-17A homodimer and IL-17AF heterodimer; b) blocking the binding of IL-17A to its receptor; and/or c) inhibiting IL-17A-mediated bioactivity.
  • the IL-17A, IL-17AF or IL-17F described herein is selected from one or more of cynomolgus monkey, mouse and human.
  • the antibody or the antigen-binding fragment thereof disclosed herein does not specifically bind to a) any one or more of human IL-17F homodimer, human IL-17B homodimer, human IL-17C homodimer, human IL-17D homodimer, and human IL-17E homodimer, and/or b) any one or more of cynomolgus monkey IL-17F homodimer and mouse IL-17F homodimer.
  • the antibody or the antigen-binding fragment thereof disclosed herein has the function of inhibiting the binding of IL-17A to its receptor, and/or reducing cell signal transduction and/or bioactivity mediated by IL-17A.
  • the antibody or the antigen-binding protein thereof disclosed herein is capable of inhibiting IL-17A from inducing the secretion of CXCL1 by epithelial cells when assessed for activity in vitro.
  • the antibody or the antigen-binding fragment thereof disclosed herein is capable of inhibiting IL-17A from inducing the secretion of CXCL1 in mice when assessed for activity in vivo.
  • the antibody or the antigen-binding protein thereof disclosed herein is capable of resisting the onset of imiquimod-induced psoriasis in the mouse model, and reducing the clinical score of the psoriasis onset in mice and the degree of ear swelling of mice, when assessed for activity in vivo.
  • the isolated antibody or the antigen-binding fragment thereof disclosed herein is capable of inhibiting knee joint swelling in antigen-induced arthritis models, such as cynomolgus monkey AIA-model, when assessed in vivo.
  • the present invention also provides the use of the antibody or the antigen-binding fragment thereof (preferably hu31, hu43, hu44, hu59, hu60 or hu250) as a medicament, preferably as a medicament for treating pathological diseases mediated by IL-17A and/or treated by inhibiting IL-17A signal transduction.
  • the antibody or the antigen-binding fragment thereof preferably hu31, hu43, hu44, hu59, hu60 or hu250
  • the pathological disease mediated by IL-17A is an inflammatory disorder or condition, such as arthritis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.
  • an inflammatory disorder or condition such as arthritis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.
  • the present invention provides a method for treating pathological diseases mediated by IL-17A, comprising administering an effective amount of the isolated antibody or the antigen-binding fragment thereof disclosed herein, preferably hu31, hu43, hu44, hu59, hu60, or hu250 antibody, so as to alleviate the condition.
  • the present invention also relates to a method for producing the antibody or the antigen-binding fragment thereof disclosed herein.
  • Such methods include isolated nucleic acid molecules encoding at least the heavy and/or light chain variable region of an antibody or an protein disclosed herein, or a cloning/expression vector comprising such nucleic acids, particularly for recombinant in a host cell to produce the antibody or the protein disclosed herein, e.g., hu31, hu43, hu44, hu59, hu60 or hu250.
  • the present invention also relates to one or more cloning vectors and a host cell comprising the expression vectors, and a method for producing the antibody or the protein comprising the antigen-binding fragment thereof disclosed herein, specifically, such as hu31, hu43, hu44, hu59, hu60 or hu250 antibody, wherein the method comprises culturing the host cell, and purifying and isolating the antibody or the protein.
  • the isolated antibody or the protein comprising an antigen-binding fragment thereof disclosed herein is conjugated to another active moiety.
  • the antibody or the antigen-binding fragment thereof disclosed herein may be a monoclonal antibody or an antigen-binding fragment thereof, preferably a chimeric antibody, a humanized antibody or a human antibody or a portion thereof.
  • composition comprising the antibody or the protein comprising the antigen-binding fragment thereof according to any of the embodiments of the present invention, in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the pharmaceutical composition comprises one or more additional active ingredients.
  • the pharmaceutical composition is a lyophilized powder. In another specific embodiment, the pharmaceutical composition is a stable liquid formulation comprising a therapeutically acceptable amount of the antibody or the molecule disclosed herein.
  • the present invention relates to the use of the antibody or the antigen-binding fragment thereof for preparing a medicament for treating any one of the pathological disorders mediated by IL-17A.
  • the present invention provides an isolated nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, an expression vector or a recombinant vector comprising the nucleic acid molecule, and a host cell transformed with the vector.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, the nucleic acid molecule, the vector or the host cell, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides the use of the antibody or the antigen-binding fragment thereof, the nucleic acid molecule, the vector or the host cell, or the pharmaceutical composition for preparing a medicament for treating and/or preventing diseases or disorders mediated by IL-17A.
  • the medicament is a medicament for treating arthritis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.
  • arthritis rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.
  • FIG. 1 shows the SDS-PAGE electropherogram of the recombinant human IL-17A-mFc protein.
  • FIG. 2 shows the binding of human IL-17A-mFc to IL-17RA on 293F cells assayed by FACS (MFI represents mean fluorescence intensity; treatment group vs. control group: 7695 vs. 308).
  • FIG. 3 shows the effect of hybridoma antibody on blocking IL-17A-mediated bioactivity in vivo assayed by ELISA.
  • the hybridoma antibody significantly inhibits IL-17A from inducing the expression of CXCL1 in mice.
  • FIG. 4 shows the binding of chimeric antibodies to human IL-17A assayed by ELISA.
  • the chimeric antibodies ch1, ch2, ch4, ch7 and ch16 bind to human IL-17A with higher specificity, with the EC 50 values being 6.62 ng/mL, 5.17 ng/mL, 88.48 ng/mL, 39.96 ng/mL and 15.42 ng/mL, respectively.
  • FIG. 5 shows the effect of chimeric antibodies on blocking the binding of human IL-17A to IL-17RA on 293F cells assayed by FACS.
  • the chimeric antibodies ch1, ch2, ch7 and ch16 block the binding efficiently, with the IC 50 values being 4.46 ⁇ g/mL, 3.145 ⁇ g/mL, 1.220 ⁇ g/mL and 1.445 ⁇ g/mL, respectively.
  • FIG. 6 shows the binding of humanized antibodies to human IL-17A assayed by ELISA.
  • the antibodies hu31, hu43, hu44, hu59, hu60 and hu250 bind to human IL-17A with higher specificity, with the EC 50 values being 8.13 ng/mL, 8.64 ng/mL, 6.764 ng/mL, 6.102 ng/mL, 5.776 ng/mL and 6.351 ng/mL, respectively.
  • FIG. 7 shows the effect of humanized antibodies on blocking the binding of human IL-17A to IL-17RA on 293F cells assayed by FACS.
  • the antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all block the binding efficiently, with the IC 50 values being 867.6 ng/mL, 780.8 ng/mL, 828.5 ng/mL, 467.4 ng/mL, 482.8 ng/mL and 577.8 ng/mL, respectively.
  • FIG. 8 shows the effect of humanized antibodies on blocking IL-17A-mediated secretion of CXCL1 by epithelial cells assayed by ELISA.
  • the antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all inhibit IL-17A from inducing the expression of CXCL1 in epithelial cells efficiently, and have stronger blocking effect than the control antibody.
  • FIG. 9 shows the effect of humanized antibodies on blocking IL-17A-mediated bioactivity in vivo assayed by ELISA.
  • the antibodies hu31, hu43, hu44, hu60 and hu250 inhibit IL-17A from inducing the expression of CXCL1 in mice efficiently, and have stronger blocking effect than the control antibody.
  • FIG. 10 A shows the effect of humanized antibody administration on the clinical score of imiquimod-induced psoriasis in mice.
  • the administration of hu31 and hu44 can significantly inhibit skin scales, induration, swelling and other conditions of inimiquimod-induced psoriasis in the mouse model, i.e., decrease the clinical score (*P ⁇ 0.05 vs. KLH).
  • FIG. 10 B shows the effect of humanized antibodies on the degree of ear swelling of mice with imiquimod-induced psoriasis.
  • the administration of hu31 and hu44 can significantly improve the degree of ear swelling. (*P ⁇ 0.05 vs. KLH, **P ⁇ 0.01 vs. KLH).
  • FIG. 11 A shows the effect of humanized antibodies on the body weight of female cynomolgus monkeys with type II collagen-induced arthritis. hu31 and hu59 improve the weight loss induced by arthritis to some extent (**P ⁇ 0.01, ****P ⁇ 0.0001, compared with “G2: vehicle group”; One-way ANOVA/Dunnett).
  • FIG. 11 B shows the effect of humanized antibodies on the score of collagen type II-induced arthritis in female cynomolgus monkeys.
  • hu31 significantly inhibits the increasing trend in clinical score of arthritis in cynomolgus monkeys (***P ⁇ 0.001, #P ⁇ 0.05, compared with G2: vehicle group; One-way ANOVA/Dunnett).
  • FIG. 12 shows the effect of humanized antibodies on NIH3T3-IL-17 cell-induced joint swelling in mice.
  • FIG. 13 shows the effect of humanized antibodies in mouse air pouch model of NIH3T3-IL-17 cell-induced inflammation.
  • the present invention relates to an antibody that specifically binds to human IL-17A and blocks IL-17A-mediated bioactivity.
  • the present invention relates to a full-length IgG antibody and an antigen-binding fragment thereof, described further below.
  • embodiments of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cytobiology, biochemistry and immunology, which are all within the skill of the art.
  • IL-17 or IL-17A is interleukin-17. Unless otherwise stated, the IL-17 usually refers to human IL-17A.
  • IL-17A expressed in vivo has an N-terminal signal peptide consisting of 23 amino acids, which is cleaved to generate mature IL-17A.
  • IL-17A disclosed herein refers to mature IL-17A, which does not comprise an N-terminal signal peptide, with an amino acid sequence set forth in SEQ ID NO: 66 and a nucleotide sequence set forth in SEQ ID NO: 67.
  • IL-17F expressed in vivo has an N-terminal signal peptide consisting of 30 amino acids, which is cleaved to generate mature IL-17F.
  • IL-17F disclosed herein refers to mature IL-17F, which does not comprise an N-terminal signal peptide, with an amino acid sequence set forth in SEQ ID NO: 68.
  • IL-17AF disclosed herein is a heterodimer of an IL-17A subunit and an IL-17F subunit, as understood by those of ordinary skill in the art.
  • immune response refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines, and complements) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship generally initiated by a protein-protein interaction such as binding of a growth factor to a receptor, resulting in transmission of a signal from one portion of a cell to another portion of a cell.
  • the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in the series of reactions causing signal transduction.
  • the penultimate process typically involves a nuclear event, resulting in a change in gene expression.
  • the term “activity” or “bioactivity”, or the term “biological property” or “biological characteristic” can be used interchangeably herein and includes, but is not limited to, epitope/antigen affinity and specificity, the ability to neutralize or antagonize IL-17A activity in vivo or in vitro, IC 50 , the in vivo stability of the antibody, and the immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of the antibody known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the targeted peptide, or with other proteins or tissues in general), and the ability to maintain high expression level of the protein in mammalian cells.
  • an “antibody” refers to any form of antibody having a desired bioactivity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.
  • an “isolated antibody” refers to the purified state of a conjugate, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium.
  • isolated(d) does not mean the complete absence of such substances or the absence of water, buffers or salts, unless they are present in amounts that will significantly interfere with the experimental or therapeutic use of the conjugates described herein.
  • a “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • a monoclonal antibody is highly specific and targets a single antigen epitope.
  • conventional (polyclonal) antibody preparations typically include a large number of antibodies targeting (or specific for) different epitopes.
  • the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as producing the antibody by any particular method.
  • a “full-length antibody” refers to an immunoglobulin molecule comprising four peptide chains when present naturally, including two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (about 25 kDa in full length) linked to each other by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of 3 domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • the VH and VL regions can be further divided into complementarity determining regions (CDRs) with high variability and more conservative regions called framework regions (FRs) that are spaced apart by the CDRs.
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs, in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant regions of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of classical complement system.
  • an “antigen-binding fragment” of an antibody (“parent antibody”) includes a fragment or a derivative of the antibody, generally including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains at least some of the binding specificity of the parent antibody.
  • binding fragments of an antibody include, but are not limited to, Fab, Fab′, F(ab′) 2 and Fv fragments; a diabody; a linear antibody; a single-chain antibody molecule, such as sc-Fv; and a nanobody and a multispecific antibody formed by fragments of the antibody.
  • a binding fragment or a derivative generally retains at least 10% of its antigen-binding activity when the antigen-binding activity is present on a molar concentration basis. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that an antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter their bioactivity (referred to as “conservative variants” or “function-conservative variants” of the antibody).
  • conjuggate refers to both an antibody and a binding fragment thereof.
  • a “single chain Fv” or “scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain.
  • an Fv polypeptide also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen-binding.
  • a “domain antibody” is an immunofunctional immunoglobulin fragment that contains only the variable region of the heavy chain or the light chain.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the 2 VH regions of the bivalent domain antibody may target the same or different antigens.
  • a “bivalent antibody” comprises 2 antigen-binding sites. In certain cases, the 2 binding sites have the same antigen specificity. However, a bivalent antibody may be bispecific.
  • a “diabody” refers to a small antibody fragment having two antigen-binding sites, which comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a “chimeric antibody” is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first and second antibodies are from different species.
  • the variable domain is obtained from an antibody of an experimental animal such as a rodent (“parent antibody”)
  • the constant domain sequence is obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject as compared to the parent rodent antibody.
  • a “humanized antibody” refers to an antibody form containing sequences from both human and non-human (such as mouse and rat) antibodies.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions (FRs) are those of a human immunoglobulin sequence.
  • the humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).
  • a “fully human antibody” refers to an antibody that comprises only human immunoglobulin sequences.
  • a fully human antibody may contain mouse glycochains if produced in mice, mouse cells, or hybridomas derived from mouse cells.
  • a “mouse antibody” refers to an antibody that comprises only mouse immunoglobulin sequences.
  • a fully human antibody may contain rat glycochains if produced in rats, rat cells, or hybridomas derived from rat cells.
  • a “rat antibody” refers to an antibody that comprises only rat immunoglobulin sequences.
  • Isotype refers to the type of antibodies (e.g., IgM, IgE, IgG such as IgG1 or IgG4) provided by the heavy chain constant region genes. Isotype also includes modified forms of one of these types in which modifications have been made to alter Fc function, for example to enhance or attenuate effector function or binding to Fc receptors.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless explicitly limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. (see U.S. Pat. No. 8,278,036 to Kariko et al., which discloses an mRNA molecule where uridine is substituted by pseudouridine, a method for synthesizing the mRNA molecule, and a method for delivering therapeutic proteins in vivo).
  • modified mRNA can be used, for example, those disclosed in U.S. Pat. No. 8,278,036 to Kariko et al. and in Patent Application No. W02013/090186A1 to Moderna.
  • a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • a “construct” refers to any recombinant polynucleotide molecule (such as plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of genomic integration or autonomous replication, comprising a polynucleotide molecule where one or more polynucleotide molecules have been linked in a functionally operative manner (i.e., operably linked).
  • the recombinant construct will typically comprise a polynucleotide of the present invention operably linked to transcription initiation regulatory sequences that will direct transcription of the polynucleotide in a host cell.
  • transcription initiation regulatory sequences that will direct transcription of the polynucleotide in a host cell.
  • Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present invention.
  • a “vector” refers to any recombinant polynucleotide construct that can be used for transformation purpose (i.e., the introduction of heterologous DNA into a host cell).
  • a “plasmid” refers to a double-stranded DNA loop into which additional DNA segments can be ligated.
  • a viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors After introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into genome of the host cell and are, thus, replicated along with the host genome.
  • certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as “expression vectors”.
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a target gene when transformed, transfected or transduced into a host cell.
  • the expression vector comprises one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host if needed.
  • IL-17A antagonist or “IL-17A blocker” refers to an antibody or an antigen-binding protein thereof that inhibits the signal transduction activity of IL-17A induced by IL-17R, thereby reducing or neutralizing IL-17A activity.
  • assays for human cells such as IL-17A dependent CXCL1 production assays for human cells. Such assays are described in more detail in the examples below.
  • activation may have the same meaning.
  • the cell or the receptor is activated, stimulated, or treated with a ligand.
  • Ligands include natural and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins, and binding compounds derived from antibodies.
  • Ligands also include small molecules, such as peptidomimetics of cytokines and peptidomimetics of antibodies.
  • Activation may refer to the activation of a cell regulated by internal mechanisms as well as external or environmental factors.
  • Response/reaction e.g., a response of a cell, a tissue, an organ, or an organism, includes changes in biochemical or physiological behaviors (e.g., concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment), where the changes are associated with an activation, a stimulation or a treatment, or are associated with an internal mechanism such as genetic programming.
  • biochemical or physiological behaviors e.g., concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment
  • the term “treat”, “treating” or “treatment” of any disease or disorder refers in one embodiment to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms).
  • “treat”, “treating” or “treatment” refers to ameliorating or improving at least one physical parameter, including those physical parameters that may not be discernible by the patient.
  • “treat”, “treating” or “treatment” refers to modulating the disease or disorder, physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both.
  • methods for assessing treatment and/or prevention of a disease are generally known in the art.
  • a “subject” includes any human or non-human animal.
  • non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cat, horse, cattle, chicken, amphibians, and reptiles.
  • cyno refers to a cynomolgus monkey.
  • “Therapeutically effective amount”, “therapeutically effective dose,” and “effective amount” as used herein refer to an amount of the IL-17A antibody or the antigen-binding fragment thereof disclosed herein that is effective for preventing or improving one or more symptoms of a disease or condition or the development of the disease or condition, when administered alone or in combination with other therapeutic agents to a cell, tissue or subject.
  • the therapeutically effective dose also refers to an amount of the antibody or the antigen-binding fragment thereof sufficient to cause an improvement in symptoms, e.g., an amount for treating, curing, preventing or improving a related condition or promoting the treatment, cure, prevention or improvement of such condition.
  • a therapeutically effective dose refers to the amount of the ingredient.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, regardless of whether these active ingredients are administered in combination, sequentially or simultaneously.
  • An effective amount of the therapeutic agent will lead to an increase in a diagnostic index or parameter by at least 10%, generally at least 20%; preferably at least about 30%, more preferably at least 40%, most preferably at least 50%.
  • Any suitable method for producing antibodies may be employed to produce the antibody disclosed herein.
  • Any suitable form of human IL-17A may be used as an immunogen (antigen) for the production of antibodies.
  • an immunogen antigen
  • any human IL-17A isotype or a fragment thereof may be used as an immunogen. Examples include, but are not limited to the natural mature human IL-17A (having an amino acid sequence set forth in SEQ ID NO: 66) as described herein.
  • the hybridoma cells producing murine monoclonal anti-human IL-17A antibodies can be produced by methods well known in the art. These methods include, but are not limited to, hybridoma techniques originally developed by Kohler et al., (1975) ( Nature 256:495-497).
  • mouse splenocytes are isolated and fused to a mouse myeloma cell line using PEG or by electrofusion according to standard schemes.
  • the resulting hybridomas producing antigen-specific antibodies can then be screened. For example, in the case of 50% PEG, a single cell suspension of splenic lymphocytes derived from an immunized mouse can be fused to 1/6 number of mouse myeloma cells SP20 (ATCC).
  • the cells can be seeded in a flat-bottomed microtiter plate at about 2 ⁇ 10 5 cells/mL, followed by 2 weeks of incubation in complete medium containing 20% fetal bovine serum and selection medium containing 1 ⁇ HAT (Sigma; HAT added 24 h after fusion). After 2 weeks, the cells can be cultured in medium with HAT replaced with HT. Wells can then be screened by ELISA for anti-human IL-17A monoclonal IgG antibodies. In general, after 10-14 days of the large-scale growth of hybridomas, the medium can be observed. The hybridomas secreting antibodies can be seeded and screened again.
  • the anti-human IL-17A monoclonal antibody hybridomas can be subcloned at least twice by limiting dilution.
  • the stable subclones can then be cultured in vitro to produce small amounts of antibodies in tissue medium for characterization.
  • the monoclonal hybridoma cells obtained in the present invention are 1F8, 2F5, and 2B2 that secrete antibodies binding to IL-17A with high specificity, blocking the binding of IL-17A to IL-17RA, and inhibiting IL-17A-mediated bioactivity, such as inhibiting the secretion of CXCL1.
  • the DNA sequences of the immunoglobulin variable regions of candidate hybridoma cells 1F8, 2F5, and 2B2 are determined using degenerate primer-based PCR in the present invention.
  • the hybridoma cell 1F8 produces two antibody light chain genes and one antibody heavy chain gene
  • 2F5 produces two antibody heavy chain genes and one antibody light chain gene. Therefore, the antibodies secreted by 1F8 and 2F5 may each include two mixed intact antibodies.
  • the antibodies secreted by 1F8 are coded as 1F8-1 and 1F8-2
  • the antibodies secreted by 2F5 are coded as 2F5-1 and 2F5-2.
  • Antibodies derived from rodents may induce unwanted immunogenicity of the antibodies when used as therapeutic agents in vivo. Repeated use of these antibodies induces an immune response in the human body to therapeutic antibodies. Such immune responses result in at least a loss of therapeutic efficacy and, at most, a potentially lethal allergic reaction.
  • One method for reducing the immunogenicity of rodent antibodies includes producing chimeric antibodies, in which the mouse variable region is fused to the human constant region (Liu et al., (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, the preservation of intact rodent variable region in chimeric antibodies can still induce deleterious immunogenicity in patients.
  • CDR complementarity determining region
  • the chimeric or humanized antibodies disclosed herein can be prepared based on the sequences of the prepared murine monoclonal hybridoma antibodies.
  • DNA encoding the immunoglobulin heavy and light chains can be obtained from a murine hybridoma of interest and engineered to comprise non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric heavy chain and the chimeric light chain can be obtained by operably linking the immunoglobulin heavy chain and light chain variable regions of hybridoma origin to human IgG constant regions respectively using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.).
  • the human IgG can be selected from any subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.
  • the chimeric antibodies disclosed herein can be obtained by “mixing and matching” a chimeric light chain expression plasmid with a chimeric heavy chain expression plasmid to transfect expression cells.
  • the binding of such “mixed and matched” antibodies to IL-17A can be assayed using the above binding assay and other conventional binding assays (e.g., ELISA).
  • the preferred ch1, ch2, ch4, ch7 and ch16 have optimal binding and blocking activity, and their amino acid sequences of variable region are shown in Table 2.
  • VH Heavy chain Light chain Chimeric variable region variable region antibody
  • VL Heavy chain Light chain Chimeric variable region variable region antibody
  • variable region CDRs in the antibodies disclosed herein may be determined using any of well-known schemes, including the Kabat scheme described by Kabat et al., (1991), Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme) and the IMGT scheme described by Lefranc M.-P. et al., (1999 Nucleic Acids Research, 27:209-212).
  • Kabat numbering scheme
  • IMGT scheme described by Lefranc M.-P. et al.
  • murine CDR regions can be inserted into human germline framework regions using methods known in the art. See U.S. Pat. No. 5,225,539 to Winter et al. and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al. Briefly, human germline IgG genes homologous to the cDNA sequence of the murine antibody variable regions were retrieved in the human immunoglobulin gene database of the NCBI (http://www.ncbi.nlm.nih.gov/igblast/) by the inventor, and in principle, humanization is achieved by grafting of the selected CDRs.
  • NCBI http://www.ncbi.nlm.nih.gov/igblast/
  • the humanization comprises the following steps: A. the gene sequences of the candidate antibodies are compared with the human germline antibody gene sequence to select sequences with high homology; B, by HLA-DR affinity analysis, a human germline framework sequence with low affinity is selected; and C, the framework amino acid sequences of the variable regions and their periphery are analyzed by computer simulation and molecular docking, and the spatial and stereo arrangement is verified.
  • the key amino acid individuals that interact with IL-17A and maintain the spatial framework in the gene sequence of the candidate antibodies are analyzed by calculating electrostatic force, Van der Waals' force, hydrophilcity and hydrophobicity, and entropy value, and grafted to the selected human germline gene framework.
  • the amino acid positions of the framework regions that must be retained are mapped. After that, the humanized antibodies are synthesized.
  • preferred humanized antibodies obtained in the present invention are hu31, hu43, hu44, hu59, hu60 and hu250.
  • Amino acid and nucleotide sequences of light/heavy chain of humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 are shown in Table 5.
  • the other antibodies disclosed herein include those having an amino acid sequence that has been mutated by amino acid deletion, insertion or substitution, but still has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or 100% identity to the above antibodies (particularly in the CDR regions set forth in the above sequences).
  • the antibody disclosed herein is a mutant of any of hu31, hu43, hu44, hu59, hu60, and hu250, wherein the mutant comprises a mutant amino acid sequence in which no more than 1, 2, 3, 4 or 5 amino acids have been mutated by amino acid deletion, insertion or substitution in a CDR region when compared to the CDR regions set forth in the above sequences.
  • nucleic acids encoding the antibody disclosed herein include those that have been mutated by nucleotide deletion, insertion or substitution, but still have at least 60%, 70%, 80%, 90%, 95% or 100% identity to the corresponding CDR coding regions set forth in the above sequences.
  • the present invention relates to one or more expression vectors or a host cell comprising the expression vectors and a method for producing the antibody or the antigen-binding fragment thereof disclosed herein, wherein the method comprises culturing the host cell, and purifying and isolating the antibody or the antigen-binding fragment thereof.
  • the antibodies disclosed herein can be produced in host expression cells using, for example, a combination of recombinant DNA technique and gene transfection method well known in the art (e.g., Morrison, S. 1985 , Science 229:1202).
  • DNA encoding partial or full-length light and heavy chains can be obtained using standard molecular biology or biochemical techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning using hybridomas expressing an antibody of interest), and the DNA can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences.
  • operably linked means that the antibody genes are linked into a vector such that the transcriptional and translational control sequences in the vector perform their intended functions of regulating the transcription and translation of the antibody genes.
  • the expression vector and expression control sequences that can be compatible with the expression host cell used are selected.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into different vectors, or more typically, both genes are inserted into the same expression vector.
  • An antibody gene is inserted into an expression vector by standard methods (e.g., ligation of the antibody gene fragment and complementary restriction sites on the vector, or blunt end ligation if no restriction site is present).
  • Full-length antibody genes of any antibody isotype can be obtained by inserting the light and heavy chain variable regions of the antibody described herein into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype to enable the operable linking of the VH segments to the CH segments in the vector and the operable linking of the VL segments to the CL segments in the vector.
  • the recombinant expression vector may encode a signal peptide (also referred to as a leader sequence) that facilitates the secretion of an antibody chain from a host cell.
  • the antibody chain gene can be cloned into a vector such that the signal peptide is linked to the amino terminus of the antibody chain gene in the same reading frame.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • Mammalian host cells for the expression of the recombinant antibodies disclosed herein include a number of immortalized cell lines available from American Type Culture Collection (ATCC). These include, in particular, Chinese hamster ovary (CHO) cells, NS0, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression level.
  • ATCC American Type Culture Collection
  • an antibody is produced by culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably, by secreting the antibody into a medium in which the host cell is grown.
  • the antibody can be isolated from the medium using standard protein purification methods. It is likely that antibodies expressed by different cell lines or in transgenic animals have different glycosylations from each other.
  • antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are integral parts of the present invention, regardless of the glycosylation of the antibody.
  • nonfucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts, and are unlikely to be immunogenic because their glycan structures are normal components of natural human serum IgG.
  • the antibody or the antigen-binding fragment thereof disclosed herein has in vitro and in vivo diagnostic and therapeutic uses.
  • the humanized antibody hu31, hu43, hu44, hu59, hu60 or hu250 is useful for treating IL-17A-related diseases or disorders.
  • the isolated antibody or the antigen-binding fragment thereof disclosed herein is capable of resisting the onset of imiquimod-induced psoriasis in the mouse model, and reducing the clinical score of the psoriasis onset in mice and the degree of ear swelling of mice, when assessed for activity in vivo.
  • the humanized antibodies hu31 and hu44 can significantly resist the onset of mice, and reduce the clinical score of the psoriasis onset in mice and the degree of ear swelling of mice.
  • the isolated antibody or the antigen-binding fragment thereof disclosed herein is capable of inhibiting knee joint swelling in antigen-induced arthritis models, such as cynomolgus monkey AIA-model, when assessed in vivo.
  • the humanized antibody hu31 significantly inhibits the increasing trend in clinical score of arthritis in cynomolgus monkeys.
  • a method for treating pathological diseases mediated by IL-17A comprising administering an effective amount of the isolated antibody or the antigen-binding fragment thereof according to the present invention, specifically hu31, hu43, hu44, hu59, hu60, or hu250 antibody, so as to alleviate the disorder.
  • the isolated antibody or the protein comprising an antigen-binding fragment thereof disclosed herein is conjugated to another active moiety.
  • the isolated antibody or the protein comprising the antigen-binding fragment thereof disclosed herein may be a monoclonal antibody or an antigen-binding fragment thereof, preferably a chimeric antibody, a humanized antibody or a human antibody or a portion thereof.
  • composition comprising the antibody or the protein comprising the antigen-binding fragment thereof according to the embodiments described herein, in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the pharmaceutical composition comprises one or more additional active ingredients.
  • the pharmaceutical composition is a lyophilized powder. In another specific embodiment, the pharmaceutical composition is a stable liquid formulation comprising a therapeutically acceptable amount of the antibody or the molecule disclosed herein.
  • the present invention provides a method for treating IL-17A-related disorders and/or autoimmune and inflammatory disorders.
  • the method comprises administering to a subject in need thereof the isolated antibody or the antigen-binding fragment thereof according to the present invention.
  • the present invention also provides a method for attenuating or inhibiting a signal transduction response induced by IL-17A or IL-17AF in a target cell or tissue by contacting a cell with a composition comprising a therapeutically effective dose of the antibody disclosed herein.
  • IL-17A-mediated disease or “IL-17A-related disorder” includes all diseases and conditions in which IL-17A or IL-17AF play a role (whether directly or indirectly), including the cause, development, progression, persistence or pathology of the disease or condition.
  • these terms include conditions associated with or characterized by abnormal IL-17A or IL-17AF level and/or diseases or conditions that can be treated by attenuating or inhibiting IL-17A/AF-induced activity (e.g., CXCL1) in a target cell or tissue.
  • diseases or conditions include inflammatory conditions and autoimmune diseases such as arthritis, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis or psoriasis.
  • diseases also include allergies and allergic conditions, hypersensitivity reactions, chronic obstructive pulmonary disease, cystic fibrosis, and organ or tissue transplant rejection.
  • inhibitor or “treat” or “treatment” includes delay in the development of symptoms associated with a disorder and/or reduction in the severity of symptoms of such disorders.
  • the term also includes ameliorating existing uncontrolled or harmful symptoms, preventing other symptoms, and ameliorating or preventing the underlying causes of such symptoms.
  • the term indicates that a beneficial result has been provided to a vertebrate subject suffering from or likely to suffer from a disorder, disease or condition.
  • therapeutically effective amount refers to an amount of an IL-17 conjugate disclosed herein that is effective for preventing or improving one or more symptoms of a disease or condition or the development of the disease or condition, when administered alone or in combination with other therapeutic agents to a cell, tissue or subject.
  • the therapeutically effective dose also refers to an amount of the conjugate sufficient to cause an improvement in symptoms, e.g., an amount for treating, curing, preventing or improving a related condition or promoting the treatment, cure, prevention or improvement of such condition.
  • a therapeutically effective dose refers to the amount of the ingredient.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, regardless of whether these active ingredients are administered in combination, sequentially or simultaneously.
  • An effective amount of the therapeutic agent will result in an increase in a diagnostic index or parameter by at least 10%, generally at least 20%; preferably at least about 30%, more preferably at least 40%, most preferably at least 50%.
  • RA Rheumatoid Arthritis
  • RA Rheumatoid Arthritis
  • RA is a progressive systemic disease characterized by inflammation of synovial joints, affecting approximately 0.5% of the global population. See Emery, (2006) BMJ 332:152-155. Inflammation of joints may lead to deformity, pain, stiffness and swelling, and ultimately to irreversible degeneration of joints. Affected joints include knee, elbow, neck and extremity joints.
  • Conventional treatments include symptomatic treatment with NSAIDs, followed by treatment with disease-modifying anti-rheumatic drugs (DMRDs), such as gold, penicillamine, sulfasalazine, and methotrexate.
  • DMRDs disease-modifying anti-rheumatic drugs
  • TNF- ⁇ inhibitors including monoclonal antibodies such as infliximab, adalimumab, and golimumab, and receptor fusion proteins such as etanercept. Treatment with such TNF-alpha inhibitors significantly reduces structural damage due to the disease.
  • the anti-IL-17A antibodies disclosed herein may be used to treat RA in a subject in need for such treatment.
  • the anti-IL-17A antibodies disclosed herein may also be combined with other treatments for RA, such as methotrexate, azathioprine, cyclophosphamide, ethyl mycophenolate, NSAIDs or TNF- ⁇ inhibitors.
  • Skin is an important barrier between the internal and external environment, preventing contact with potentially harmful antigens.
  • T cells, polymorphonuclear cells, macrophages and the like at the skin contact site locally infiltrate and an inflammatory response is initiated to eliminate the antigen.
  • this inflammatory response triggered by the pathogen is under strict control and is terminated when the pathogen is eliminated.
  • such inflammatory response occurs without external irritation and without proper control, resulting in skin inflammation.
  • the present invention provides a method for treating and diagnosing such skin inflammation.
  • Skin inflammation (a consequence of the aforementioned cellular infiltration and cytokines secreted by the cells) includes several inflammatory conditions such as cicatricial pemphigoid, scleroderma, hidradenitis suppurativa, toxic epidermal necrolysis, acne, osteitis, graft versus host disease (GVHD), pyroderma gangrenosum and Behcet's syndrome (see, e.g., Williams and Griffiths, (2002) Clin. Exp. Dermatol., 27:585-590).
  • the most common skin inflammation is psoriasis.
  • Psoriasis is characterized by T cell-mediated keratinocyte hyperproliferation with inflammatory infiltration.
  • the disease has some shared manifestations, including chronic plaque lesions, rash, and pustular lesions (see, e.g., Gudjonsson et al., (2004) Clin. Exp. Immunol. 135:1-8). About 10% of patients with psoriasis may develop arthritis. The disease has a strong and complicated genetic predisposition with a 60% identity in monozygotic twins.
  • Typical psoriatic lesions are red plaques with clear edge covered by thick silvery scales. Inflammation and hyperproliferation of psoriatic tissue are associated with different histological, antigenic and cytokine profiles compared to normal skin. Cytokines associated with psoriasis are: TNF- ⁇ , IL-19, IL-18, IL-15, IL-12, IL-7, IFN- ⁇ , IL-17A and IL-23 (see Gudjonsson et al., supra).
  • anti-IL-17A antibodies disclosed herein may also be used to prevent, treat, diagnose and predict psoriasis onset.
  • the antibody is mixed with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984).
  • the following dosage forms of therapeutic and diagnostic agents may be prepared by mixing the antibody with an acceptable carrier, excipient or stabilizer: a lyophilized powder, a paste, an aqueous solution or a suspension.
  • an acceptable carrier excipient or stabilizer
  • a lyophilized powder e.g., a paste, an aqueous solution or a suspension.
  • the IL-17A antibody disclosed herein is diluted to an appropriate concentration by a sodium acetate solution (pH 5-6), and added with NaCl or sucrose to adjust osmolality.
  • Other substances e.g., polysorbate 20 or polysorbate 80 may be added to improve stability.
  • the dosage regimen depends on several factors including serum or tissue turnover of the therapeutic antibody, severity of symptoms, immunogenicity of the therapeutic antibody, and accessibility of the target cells in the biological matrix.
  • the dosage regimen delivers a sufficient amount of therapeutic antibody to achieve an improvement in the target disease state while minimizing adverse side effects.
  • the amount of biologics delivered depends in part on the particular therapeutic antibody and the severity of the condition to be treated.
  • Guidance regarding selections of appropriate dosages for therapeutic antibody is available. Appropriate dosages can be determined by the clinician, for example, using parameters or factors known or suspected in the art affecting treatment. In general, the dosage starts at an amount slightly less than the optimal dose and will be increased in small increments thereafter until the desired or optimal effect is achieved relative to any negative side effects.
  • Important diagnostic methods include, for example, diagnostic methods based on inflammatory symptoms or levels of inflammatory cytokines produced.
  • biologics derived from the same species as the animal for the targeted therapy are used, thereby minimizing inflammatory, autoimmune or proliferative responses to the agent.
  • chimeric, humanized and fully human antibodies are preferred.
  • the present invention includes any combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of applicability will become apparent from the detailed description provided below. However, it should be understood that the detailed description and the specific examples, while indicating preferred embodiments of the present invention, are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description. All publications, patents and patent applications cited herein, including the citations, are hereby incorporated by reference in their entirety for all purposes.
  • Plasmid HG12047-G containing the cDNA sequence encoding full-length human IL-17A (NCBI Accession No. NP_002181.1) was purchased from Sino Biological, Inc., and a mature fragment of human IL-17A (amino acids 24-155 of NCBI Accession No. NP_002181.1, amino acid sequence of SEQ ID NO: 66, nucleotide sequence of SEQ ID NO: 67) was amplified by conventional PCR techniques.
  • the amplified fragment was digested by BSPQI, and was cloned into a eukaryotic expression plasmid system (MXT1-Fc, containing the Fc domain of murine IgG heavy chain) constructed in-house, thereby generating an expression plasmid for recombinant fusion protein IL-17A-mFc.
  • Verified plasmid was transfected into the expression cell 293F by conventional techniques, and the recombinant human IL-17A-mFc protein was acquired after expression and purification.
  • FIG. 1 shows the SDS-PAGE electropherogram of the recombinant human IL-17A-mFc protein.
  • Plasmid HG10895-G containing the cDNA sequence encoding full-length human IL-17RA was purchased from Sino Biological, Inc., and the DNA sequence encoding full-length human IL-17RA was amplified by conventional PCR (SEQ ID NO: 69). The amplified fragments were cloned into a eukaryotic expression plasmid system (HXP) constructed in-house containing puromycin screening system by conventional cloning techniques. Verified recombinant expression plasmid for IL-17RA was transfected into 293F cells (ATCC). 24 h after transfection, cells were selected by puromycin (2 ⁇ g/mL) until stable IL-17RA 293F cells were generated.
  • HXP eukaryotic expression plasmid system
  • IL-17RA-293F monoclones were selected and passaged to give stable IL-17RA 293F cell lines. All clones were screened by FACS or the like, and clones with top expression level were selected by FACS binding assay for hybridoma monoclonal antibodies or for use in functional assays.
  • the binding specificity of the recombinant IL-17A-mFc protein to IL-17RA on 293F cells was determined by FACS. Briefly, cells (stable IL-17RA 293F cell lines) were prepared into cell suspensions at 1 ⁇ 10 6 /mL, and added to 96-well plates at 20 ⁇ L/well, with an actual cell number of 2 ⁇ 10 4 /well. The recombinant IL-17A-mFc protein (3 ⁇ g/mL, 20 ⁇ L/well; treatment group) or 1% BSA (20 ⁇ L/well; negative control group) was mixed with the cell suspension, and after an incubation at 37° C. for 30 minutes, the cells were washed with FACS buffer 3 times.
  • Anti-mouse IgG (1:200) was added and the cells were incubated at room temperature for 30 minutes. The cells were washed 3 times with FACS buffer and detected by a flow cytometer to compare the mean fluorescence intensity (MFI) of the groups. As shown in FIG. 2 , the recombinant IL-17A-mFc protein can specifically bind to IL-17RA on 293F cells.
  • Hybridoma antibodies were produced using standard molecular biological techniques. Briefly, native human IL-17A protein purchased from HumanZyme as an antigen was mixed with an equal amount of an immunoadjuvant. 5 female FVB mice aged 6 weeks were immunized. One booster immunization was performed weekly after the primary immunization, totaling seven immunizations. After the last booster immunization, mice with high anti-IL-17A antibody titers in serum were selected for cell fusion. Spleen cells were isolated and fused with the murine myeloma SP2/0 cells (ATCC) by standard hybridoma techniques. The fused cells were resuspended in complete medium RPMI-1640 containing HAT and plated in wells with a feeder layer of peritoneal cells.
  • Monoclonal hybridoma secretory supernatants were identified based on initially desired antibody/antigen binding characteristics (such as binding affinity for IL-17A, ability to block the binding of IL-17A to its receptor, cross-reactivity, and ability to block IL-17A-mediated biological effects in vitro).
  • Antibodies in supernatant from hybridomas 1F8, 2B2, 2F5, 2F2, 2H1 and 2H5 were used for further characterization.
  • IL-17A promotes the expression and release of cytokine CXCL1 in vivo.
  • the expression change of CXCL1 in mouse serum can be quantitatively detected through ELISA, so as to determine the influence of a hybridoma antibody on IL-17A-mediated bioactivity in mice.
  • 40 female Balb/c mice aged 10 weeks were selected and divided into 8 groups of 5 mice each. 4 days before administration, serum was collected and CXCL1 expression was measured as the baseline.
  • candidate hybridoma antibodies, saline (control) or reference antibody mAb317 commercially available anti-IL-17A antibody, from R&D was administered intracardially at a dose of 1 mg/kg.
  • the hybridoma antibodies obtained in Example 4 and the commercial antibody mAb317 both can significantly inhibited IL-17A from inducing CXCL1 expression in mice.
  • hybridomas 1F8, 2B2 and 2F5 The DNA sequences of the antibody variable regions expressed by hybridomas 1F8, 2B2 and 2F5 were determined using degenerate primer-based PCR. Briefly, hybridoma cell lines 1F8, 2B2 and 2F5 were expanded and harvested by centrifugation at 1000 rpm. Total RNA was extracted using Trizol. Using the total RNA as a template, a first-strand cDNA was synthesized. DNA sequences of corresponding variable regions were amplified by PCR using the first-strand cDNA as a subsequent template. The PCR primers used were based on an Ig-primer set. PCR products were collected, purified, sequenced and analyzed to give the variable region sequences of the heavy and light chains of the candidate hybridoma antibodies.
  • the hybridoma cell 1F8 produced two antibody light chain variable region gene sequences and one antibody heavy chain variable region gene sequence
  • 2F5 produced two antibody heavy chain variable region gene sequences and one antibody light chain variable region gene sequence. Therefore, the antibodies secreted by 1F8 and 2F5 might each include two intact antibodies, wherein the antibodies secreted by 1F8 were coded as 1F8-1 and 1F8-2, and the antibodies secreted by 2F5 were coded as 2F5-1 and 2F5-2.
  • amino acid sequences of the heavy and light chain variable regions of the antibodies expressed by 1F8, 2F5, 2B2 are shown in Table 1 (see DETAILED DESCRIPTION).
  • Human IgG4 heavy chain constant region Fc fragment and light chain kappa constant region were cloned from human blood cells (Beijing Blood Institute) and ligated with pCDNA3.1 plasmid for engineering.
  • the heavy chain and light chain variable region sequence fragments were synthesized by GenScript.
  • the heavy and light chains were cleaved by Bspq I, and then ligated to a correspondingly modified pCDNA3.1 plasmid.
  • the expression plasmids of IgG4 chimeric heavy chain (ch-HC) or light chain (ch-LC) were verified by sequencing.
  • the expression plasmids for the different chimeric heavy and light chains were paired to transfect expression cells, producing 16 chimeric antibodies numbered ch1 to ch16 (see Table 6). All subsequent experimental materials were given by the expression cells transfected with these plasmids.
  • the binding specificity of the chimeric antibodies to human IL-17A was determined by conventional ELISA. Namely, 0.5 ⁇ g/mL of human IL-17A-mFc was immobilized on a 96-well plate and incubated for 60-90 minutes at 37° C. The solution in wells was then discarded, and the wells were washed 3 times with washing buffer, and blocked for 60 minutes with PBS containing 2% BSA. The plate was washed 3 times with washing buffer, and added with diluted chimeric antibodies at different concentrations. The antibodies were incubated at 37° C. for 60 minutes. The plate was then washed 3 times with washing buffer, and added with 10000-fold diluted biotinylated anti-IgG4 antibody.
  • the system was incubated at 37° C. for 1 hour, washed three times with washing buffer, added with HRP-Strep 10000-fold diluted with washing buffer, and incubated at room temperature for 1 hour. After 3 washes with washing buffer, 100 ⁇ L of TMB substrate was added for color development. The system was incubated at room temperature for 30 minutes, and the reaction was terminated with 100 ⁇ L of 2 M hydrochloric acid solution. The absorbance at 450 nm was measured by a plate reader.
  • chimeric antibodies ch1, ch2, ch4, ch7 and ch16 bind to human IL-17A with higher specificity, with the EC 50 of the chimeric antibodies being 6.62 ng/mL, 5.17 ng/mL, 88.48 ng/mL, 39.96 ng/mL and 15.42 ng/mL, respectively.
  • the blocking of binding of IL-17A to IL-17RA on cells was detected by a competitive cell-based flow cytometry (FACS) assay. Briefly, chimeric antibody dilutions of different concentrations (3-fold diluted from 10 ⁇ g/mL) were mixed with the pre-biotinylated human IL-17A-mFC (3 ⁇ g/mL) obtained in Example 1, and incubated at room temperature for 30 minutes. The mixture was then co-incubated with a cell suspension (stable IL-17RA 293F cell lines obtained in Example 2, 1.5 ⁇ 10 5 cells/well) at 37° C. for 15 minutes.
  • FACS flow cytometry
  • chimeric antibodies ch1, ch2, ch7 and ch16 can significantly inhibit the binding of human IL-17 to IL-17RA on the surface of 293F cells. Combining other experimental results, ch1 and ch16 were selected for further humanization.
  • human IgG genes homologous to the cDNA sequence of the murine antibody variable regions were retrieved in the human immunoglobulin gene database of the NCBI (http://www.ncbi.nlm.nih.gov/igblast/). The amino acid sequences of CDRs of the variable regions and their boundaries were then determined as per the Kabat numbering system or the IMGT numbering system. Human IGVH and IGVk with high homology to the variable regions of the murine antibody were selected as templates for humanization, and were humanized by CDR grafting. Briefly, the humanization comprises the following steps: A. the gene sequences of the candidate antibodies were compared with the human germline antibody gene sequence to select sequences with high homology; B.
  • a human germline framework sequence with low affinity was selected; and C. the framework amino acid sequences of the variable regions and their periphery were analyzed by computer simulation and molecular docking, and the spatial and stereo arrangement was verified.
  • the key amino acid individuals that interact with IL-17A and maintain the spatial framework in the gene sequence of the candidate antibodies were analyzed by calculating electrostatic force, Van der Waals' force, hydrophilicity and hydrophobicity, and entropy value, and grafted to the selected human germline gene framework. The amino acid positions of the framework regions that must be retained were mapped. After that, the humanized antibodies were synthesized.
  • variable region CDRs of murine antibodies and their amino acid sequences are shown in Table 3 of the DETAILED DESCRIPTION.
  • chimeric antibodies ch1 and ch16 were selected for humanization. Following a primary screening of a series of antibody/antigen-binding properties (such as binding affinity for IL-17A, ability to block the binding of IL-17A to its receptor), humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 were selected for subsequent validation.
  • a series of antibody/antigen-binding properties such as binding affinity for IL-17A, ability to block the binding of IL-17A to its receptor
  • humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 were selected for subsequent validation.
  • humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 specifically bound to IL-17A.
  • the EC 50 was 8.13 ng/mL, 8.64 ng/mL, 6.76 ng/mL, 6.10 ng/mL, 5.78 ng/mL and 6.35 ng/mL, respectively.
  • IL-17A The blocking of binding of IL-17A to IL-17RA on cells was detected by a competitive cell-based flow cytometry (FACS) assay.
  • FACS flow cytometry
  • the method and procedures are described in Example 9.
  • humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 significantly inhibited IL-17A from specifically binding to IL-17RA on cells.
  • the IC 50 was 867.6 ng/mL, 780.8 ng/mL, 828.5 ng/mL, 467.4 ng/mL, 482.8 ng/mL and 577.8 ng/mL, respectively.
  • IL-17A can stimulate the expression and release of a cytokine CXCL1 in various epithelial cells and other cells.
  • the change in expression level of CXCL1 in cell supernatant can be quantified by ELISA to determine the influence of the humanized antibodies on the bioactivities mediated by IL-17A in the cells.
  • HT-29 cells human colorectal adenocarcinoma epithelial cells, ATCC
  • ATCC human colorectal adenocarcinoma epithelial cells
  • Dilutions (3-fold serial dilution from 55 ⁇ g/mL) of humanized antibodies hu31, hu59, hu60, hu250 or a reference antibody (secukinumab, Novartis) were mixed with human IL-17A (1 ⁇ g/mL), transferred to 96-well plates, and incubated for 1 h.
  • 100 ⁇ L of suspensions (2 ⁇ 10 4 cells) of HT-29 cells ATCC, human colorectal adenocarcinoma epithelial cells
  • the cells were centrifuged at 500 ⁇ g for 5 min, and the supernatant was transferred to new 96-well plates.
  • the CXCL1 expression was determined by an ELISA kit. As shown in FIG. 8 , the humanized antibodies hu31, hu59, hu60 and hu250 demonstrated more potent antagonism to IL-17A stimulation of CXCL1 release in epithelial cells than the reference antibody secukinumab.
  • humanized antibodies on IL-17A-mediated bioactivity in vivo was determined by measuring changes in mouse serum CXCL1 levels as described in Example 5. Briefly, 40 female Balb/c mice aged 10 weeks were selected and divided into 8 groups of 5 mice each. 4 days before administration, serum was collected and CXCL1 expression was measured as the baseline. On the day of administration, candidate antibodies (humanized antibodies hu31, hu43, hu44, hu60 and hu250), or an IgG4 isotype control (hIgG) was administered intracardially at a dose of 1 mg/kg.
  • candidate antibodies humanized antibodies hu31, hu43, hu44, hu60 and hu250
  • IgG4 isotype control hIgG
  • the candidate humanized antibodies hu31, hu43, hu44, hu60 and hu250 demonstrated more potent antagonism to IL-17A stimulation of CXCL1 release in epithelial cells than the IgG4 isotype control.
  • Imiquimod administered on the ear and back skins of a mouse can induce psoriasis-like pathological characteristics, including keratinocyte hyperproliferation, inflammatory cell aggregation, dermal papillary vascular hyperplasia and the like, thus constructing a mouse model with psoriasis.
  • the efficacy of the antibodies on mice with psoriasis was determined by endpoints such as clinical scores, ear swelling degrees and the like.
  • mice in group II-VI were administered with approximately 62.5 mg of imiquimod cream (Aldara, 5%, 3M Health Care Limited) on the right ear and back skins for 4 consecutive days.
  • the thickness of the right ear of the mouse was measured daily by a micrometer starting from the day of sensitization. With the ear thickness one day 1 as the baseline, the ear swelling thickness was recorded. The mice were weighed daily, observed for skin scales, induration and erythema, and classified by a 4-score scale: 0, none; 1, mild; 2, moderate; 3, severe; 4, serious. Results were recorded in mean ⁇ standard error (mean ⁇ SEM). Difference between groups were identified by one-way analysis of variance (ANOVA) and validated by Student's t test, with a P ⁇ 0.05 indicating significant difference.
  • ANOVA analysis of variance
  • the administration of the candidate antibodies disclosed herein can significantly inhibit skin scales, induration, swelling and other conditions of imiquimod-induced psoriasis in the mouse model, demonstrating decreased scores.
  • imiquimod cream administered to mouse right ear from the day of sensitization induced severe swelling and increased ear thickness in the right ear, while the humanized antibody of the present invention significantly improved the ear swelling.
  • the humanized antibody disclosed herein can obviously resist the morbidity in mice, demonstrating reductions in clinical score of the mice and the ear swelling degree.
  • RA rheumatoid arthritis
  • Such models have similar histopathological features as human RA, and are characterized by inflammation in facet joints and progressive erosion in cartilages and bones.
  • the human/humanized biological macromolecules including antibodies usually have better cross reactivity with antigens from cynomolgus monkey.
  • the cynomolgus monkey model with arthritis is an effective system for analyzing the anti-rheumatism efficacy of the humanized antibody IL-17A disclosed herein.
  • the experiment evaluated the efficacy of the candidate antibodies in a cynomolgus monkey model with arthritis.
  • Bovine collagen type II (CII, Sichuan University) was dissolved in acetic acid (CAT #10000218; Sinopharm, Shanghai, China), stirred overnight in a 4° C. refrigerator, and emulsified with an equal volume of complete Freund's adjuvant (CAT #F5881, Sigma-Aldrich, USA) to give a collagen emulsion with a final concentration of 2 mg/mL.
  • the animals were anesthetized with Zoletil (1.5-5 mg/kg, i.m.), and administered with the collagen emulsion at multiple sites on the back and tail, with 1.5%-5% isoflurane to maintain the anesthetic effect as needed.
  • the animals were re-administered with collagen 3 weeks later (day 21) with the same method as before.
  • the animals were then divided into 4 groups.
  • G1 was normal group without arthritis induction;
  • G2 was vehicle control group;
  • G3 was antibody hu31 treatment group;
  • G4 was antibody hu59 treatment group.
  • Animals with a score being 5% of the maximum clinical score (192 ⁇ 5% ⁇ 10) were sequentially divided into the groups until all animals meeting the requirement were allocated. After the allocation, the treatment was administered at 7.5 mg/kg by infusion pump in 30 minutes, once weekly for 5 weeks.
  • Body weight The body weight of the animals was measured the day before arthritis induction and once weekly thereafter until the end of the study.
  • Arthritis scoring the monkeys were classified for inflammation on days 0 and 21, and once weekly after day 21 until the end of the study (if early onset occurred, the weekly scoring should start accordingly). The scoring criteria are shown in Table 2. 15 joints in each paw were assessed: 5 metacarpophalangeal joints (MCP), 4 proximal interphalangeal joints (PIP), 5 distal interphalangeal joints (DIP), 1 wrist joint and 1 ankle joint. It is also desirable to assess the severity of lesions in knee/elbow joints in extremities. The sum of the individual joint scores was the arthritis score for the animal, with a maximum score of 192 (16 ⁇ 3 ⁇ 4).
  • Scoring criteria for arthritis 0, normal; 1, mild arthritis, with slight but distinguishable lesion; 2, moderate swelling; 3, severe arthritis, with severe swelling or obvious joint deformity.
  • Experimental data are presented in mean ⁇ standard error (mean ⁇ S.E.M). Differences in parameters between vehicle control group, reference drug group and test article group were summarized, with a p ⁇ 0.05 indicating statistically different (One-way ANOVA/Dunnett).
  • G1 the body weights of cynomolgus monkeys in the normal group (G1) were stable; for the cynomolgus monkeys with induced arthritis, the average weight in the vehicle control group (G2) continuously decreased, and the decreases in monkeys receiving antibodies hu31 and hu59 were controlled.
  • hu31 and hu59 improve the weight loss caused by arthritis to some extent (**P ⁇ 0.01, ****P ⁇ 0.0001, compared with “G2: vehicle group”; One-way ANOVA/Dunnett).
  • the arthritis clinical scores of animals in normal group remained 0 after allocation; the arthritis score of animals in the vehicle control group (G2) was progressively increased, while the test antibodies hu31 and hu59 significantly inhibited the increasing trend of the arthritis clinical score.
  • antibodies hu31 and hu59 demonstrated inhibitory effects on the progression of arthritis, and hu31 significantly inhibited the increasing trend in clinical score of arthritis in cynomolgus monkeys (***P ⁇ 0.001, #P ⁇ 0.05, compared with G2: vehicle group; One-way ANOVA/Dunnett).
  • Cells NIH3T3 cells, NIH3T3 cells expressing human IL-17.
  • NIH3T3-IL-17+ reference IgG antibody group (30 mg/kg);
  • NIH3T3-IL-17+ treatment antibody high-dose group (hu31, 3 mg/kg);
  • NIH3T3-IL-17+ treatment antibody medium-dose group (hu31, 10 mg/kg);
  • NIH3T3-IL-17+ treatment antibody low-dose group (hu31, 30 mg/kg);
  • NIH3T3-IL-17+ positive drug group (Cosentyx, 10 mg/kg).
  • NIH3T3-IL-17 cells and NIH3T3 control cells were injected into the right ankle cavity of the mice.
  • Intraperitoneal injections of the antibody hu31 (3, 10, 30 mg/kg) and Cosentyx (10 mg/kg) were given once every 3 days, starting from 1 day before model selection.
  • the efficacy of antibody hu31 injection in the mouse model with arthritis was examined.
  • the ankle thickness was measured by a vernier caliper, and the swelling extent was calculated.
  • mice As shown in FIG. 12 , after NIH3T3 cells expressing hIL-17 were injected into the joint cavity of mice, and severe swelling of mice joint was observed the next day.
  • inhibition rate (%) (ankle thickness of NIH3T3-IL-17 IgG group—ankle thickness of treatment group)/(ankle thickness of NIH3T3-IL-17 IgG group—ankle thickness of NIH3T3 group) ⁇ 100.
  • the inhibition rate of swelling by Cosentyx (10 mg/kg) was 67.1%.
  • the average inhibition rates were calculated on different days in the treatment groups and the results showed that the average inhibition rates were 45.2%, 57.0% and 73.9% for 3, 10 and 30 mg/kg treatment groups, respectively.
  • the average inhibition rate of swelling by Cosentyx (10 mg/kg) was 60.4%.
  • the results suggest that antibody hu31 can inhibit IL-17-induced ankle swelling in mice dose-dependently, and the efficacy at 10 mg/kg is equivalent to that of a positive drug Cosentyx (10 mg/kg).
  • the efficacy at 30 mg/kg is superior to that of the reference drug Cosentyx (10 mg/kg).
  • Example 18 Efficacy of Humanized Antibodies in Mouse Air Pouch Model of NIH3T3-IL-17 Cell-Induced Inflammation
  • Cells NIH3T3 cells, NIH3T3 cells expressing human IL-17.
  • NIH3T3-IL-17+ reference IgG antibody group (30 mg/kg);
  • NIH3T3-IL-17+ treatment antibody high-dose group (hu31, 30 mg/kg);
  • NIH3T3-IL-17+ treatment antibody medium-dose group (hu31, 10 mg/kg);
  • NIH3T3-IL-17+ treatment antibody low-dose group (hu31, 3 mg/kg);
  • Air pouch The mice were injected with 2.5 mL of air on the back on days 0 and 3. Starting from day 5, cells were injected into the air pouch. Each mouse received 2 ⁇ 10 5 cells/500 ⁇ L PBS. The mice were allocated into groups each containing 8 mice.
  • Leukocytes migrated into the air pouch. Cells in lavage fluid was counted, and the proportion of Gr1 + cells was determined by flow cytometry to calculate the number of neutrophils.
  • mice with air pouch were administered with NIH3T3 cells expressing hIL-17A
  • the number of infiltrating leukocytes in the air pouch of the NIH3T3-IL-17 group significantly increased, and the proportion and number of Gr1 + cells were also significantly elevated, indicating a successful model selection.
  • the antibody hu31 (at 3, 10 and 30 mg/kg) was intraperitoneally administered.
  • inhibition rate (%) (cell count of NIH3T3-IL-17-IgG group—cell count of treatment group)/(cell count of NIH3T3-IL-17-IgG group—cell count of NIH3T3 group) ⁇ 100.
  • the results showed that the total infiltrating cell count in the treatment groups of antibody hu31 (3, 10 and 30 mg/kg) was inhibited by 50.0%, 56.7% and 78.3%, respectively.
  • the inhibition rate of the Gr1 + cell count was calculated, demonstrating that: the average Gr1 + cell inhibition rates of all treatment group with antibody hu31 (3, 10 and 30 mg/kg) were 59.1%, 54.5% and 81.8%, respectively.
  • Antibody hu31 can inhibit IL-17 induced total cell infiltration and inflammatory cell infiltration in mice in a dose-dependent manner.

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